Regulated proteolysis by the ubiquitin-proteasome system (ubiquitin system) plays essential roles in a multitude of biological processes, and has major ramifications for human health. Our studies of the ubiquitin system and the N-end rule pathway over the last three decades were made possible, to a large extent, by the present grant (DK039520), currently in its 27th year of support. This renewal application is about the N-end rule pathway of protein degradation. In eukaryotes, this pathway is a specific part of the ubiquitin system. The N-end rule relates the in vivo half-life of a protein to the identity of its -terminal residue. The N-end rule pathway of protein degradation consists of two distinct branches, the Ac/N-end rule pathway and the Arg/N-end rule pathway. The Ac/N-end rule pathway was discovered by our laboratory in 2010. It recognizes proteins with N-terminally acetylated residues. The Arg/N-end rule pathway, which targets specific unacetylated N-terminal residues, was discovered by our laboratory in 1986. This pathway continues to be a fount of biological insights and remains a major focus of our studies. The present (DK039520) renewal application is about the Arg/N-end rule pathway and the identification (as well as detailed studies) of new physiological Arg/N-rule substrates. Therefore the previous title of the DK039520 grant (Ubiquitin Ligases, Mechanisms and Functions of the N-End Rule Pathway) was modified as follows in the present renewal application, to reflect its current emphasis: The Mammalian Arg/N-End Rule Pathway: Substrates, Functions, and Mechanisms. The projects of this renewal application include functional and mechanistic studies of specific physiological substrates of the mammalian (mouse) Arg/N-end rule pathway, and the identification of new mammalian Arg/N-end rule substrates, including those that act as proapoptotic protein fragments, i.e., fragments whose formation during apoptosis (a specific kind of programmed cell death) increases the probability of apoptosis.